This proposal will address fundamental questions concerning the structure of the mammalian nucleus and its relationship to pre-mRNA metabolism. The research seeks to elucidate the functional significance of non-nucleolar compartments enriched in RNA metabolic factors from a perspective which emphasizes integration with the higher-level organization of genes and chromosomes. The focus will be on the relationship of specific gene transcription and splicing to a compartment comprised of approximately 20- 40 discrete domains highly enriched in splicing factors and poly A RNA, referred to here as SC-35 domains. During the current funding period we demonstrated that specific genetic loci associate with both SC-35 domains and coiled bodies, demonstrating the integration of genomic sequences with these compartments. Using fluorescence in situ hybridization techniques largely developed in this lab, this study will investigate key aspects of the overall hypothesis that some specific active genes associate in an expression dependent manner with domains enriched in RNA metabolic factors, in which multiple functions related to pre-mRNA metabolism likely occur. The potential sub-compartmentalization of individual domains relative to the precise localization of col 1A1 transcription, splicing, and transport, will be examined in both normal cells and cells from patients with Osteogenesis Imperfecta (OI). The impact of the splice mutant on nuclear RNA distribution and "tracks" will be examined. This work will determine whether multiple genes associate with individual domains, and simultaneously explore if related genes or genes on the same chromosome associate with common domains. Since not all active genes associate with the SC-35 rich compartment, several studies will examine and better define the relationship between transcription and nuclear compartmentalization. As transcribed exogenous sequences have been shown to associate with domains, this study will investigate the possibility that DNA or RNA sequences can be targeted to pre-existing cellular domains, and will investigate whether domain association affects the level of expression. An important consideration in our experimental designs, which will be investigated is the possibility that the chromosomal context of the gene impacts its compartmentalization, potentially contributing to effect of chromosomal position on expression. The fundamental nature of this work gives it far-reaching implications for understanding vital nuclear functions. It further has potential for diagnosing and elucidating the molecular pathology of splicing defects in OI Type I and other genetic diseases.